BAF_Protocol_012_Lipidomics LC-MS(/MS): Vanquish UPLC and Orbitrap ID-X

Abstract

This protocol is the basic LC and MS running parameters for lipids or very hydrophobic molecules. Most are singly charged. Lipids can be more challenging as more mass isoforms exist.

Steps

Prepare samples for injection

Add 10 uL of Splash Mix standard to the extracted lipids. Dry samples under liquid nitrogen at 40C.

Suspend dried extracted samples with 100µL of 1:1 IPA/ACN

Mix well using a “figure- eight ” movement of the vials in the bench. Transfer the sample to a new vial with borosilicate inserts.

Make sure there is no air in the bottom of the inserts add the vials into the autosampler of the UPLC Vanquish system.

In the sequence setup view of the Xcalibur software include each sample in a row filling with: file name, sample ID, folder directory to save results, directory for the acquisition method, vial position and injection volume. Normally use 10% of each sample for injection.

LC parameters 15 min gradient

Speed limits: draw speed 1 uL/s; dispense speed 1 uL/s.

Injection wash parameters: Wash mode: both; Wash time: 10.0s; Wash speed: 10.0 uL/s.

Column temperature:55°C

Run time: 0h 33m 0s

Solvents: %A1: 60% ACN in 10 mM Ammonium Formate with 0.1%FA.

%A2: 90% IPA, 10% ACN, 10 mM Ammonium Formate with 0.1% FA.

Upper limit pressure: 1500 bar

Flow = 0.260 ml/min

Equilibration: -3min, 32% B, curve = 5.

Gradient (all steps curve = 5): 0 -1.5 min 32%B; 4 min 45% B; 5 min 52% B; 8 min 58% B; 11min 66% B; 14min 70%B; 18 min 75%B; 21min 97%B; 25 min 97%B; 25.01 min 32% B, 25.01-33 min 32%B.

General Instrument (Orbitrap ID-X) – Positive and Negative modes

Ion Source Type: H-ESI

Spray Voltage Positive 3500V

Spray Voltage Negative 2500V

Ion Transfer Tube Temperature Temperature 275°C , Sheath gas 35, Aux Gas 7, Sweep gas 1, Vaporizer Temp Temperature 350°C

Default charge state: 1

Full scan parameters

Scan 200-2000 m/z, resolution 120K, Quadrupole isolation: True, normalized AGC 50%, RF lens 40%, Max. Inj. Time 50ms, 1 microscan, Data Type Profile, Polarity Positive or Negative (create two methods one for Positive acquisition and another for Negative acquisition)

MS/MS parameters

Min intensity 5E4, Dynamic exclusion - 1 time, 5s, 10ppm, isolation 1.5 m/z, resolution 15K, HCD NCE stepped 25, 30, 35%, Max. Inj. Time 50ms, normalized AGC 50%, 1 microscan, Data Type Centroid.